Chronic otitis media with and without cholesteatoma can lead to conductive as well as sensorineural hearing loss as the result of inflammatory bone resorption in the middle ear. Conductive hearing loss is commonly caused by erosion of the ossicles resulting from the inappropriate activation of osteoclasts. A number of factors regulate osteoclast development and activation including hormones, growth factors and cytokines. Recently, nitric oxide (NO) has been shown to play a role in osteoclast activation. NO is a short-lived neutral free radical gas synthesized from L-arginine by the enzyme nitric oxide synthase (NOS). The mechanism by which nitric oxide mediates osteoclast function is not well characterized and the three known isoforms may have opposing effects. In neurons, NOS I has extremely complex transcriptional and translational regulatory mechanisms involving multiple alternate splice variants, which are expressed in a stage and tissue specific manner. Preliminary results in our laboratory demonstrate NOS I-/ -osteolcasts have an in vitro and in vivo phenotype, suggesting NOS I plays an important role in osteoclast function and that this role may be mediated by a unique splice variant of this enzyme. The specific aims of this proposal are 1) to identify the alternate splice variant(s) expressed in osteoclasts and 2) to initiate the characterization of the translated NOS I protein. In addition to providing a understanding of the molecular mechanisms underlying cholesteatoma-induced bone resorption, the identification and characterization of unique transcriptional and translational NOS I regulatory mechanisms may facilitate the discovery of novel, osteoclast-specific pharmaceutical targets.